Myxovirus Resistance A Research Articles

IL-17A but not IL-22 suppresses the replication of hepatitis B virus mediated by over-expression of MxA and OAS mRNA in the HepG2.2.15 cell line

Interleukin-17A (IL-17A) and interleukin-22 (IL-22), mainly secreted by interleukin-17-producing T help cells (Th17), are pleiotropic cytokines that regulate the biological responses of several target cells, including hepatocytes. Th17 frequency was reported to negatively correlate with plasma hepatitis B virus (HBV) DNA load in patients with HBV infection. Several studies have indicated that cytokines, such as IL-6 and IL-4, are involved in the noncytopathic suppression of HBV replication. We therefore hypothesized that IL-17A and IL-22 might have a potent suppressive effect on HBV replication. In our present study, we analyzed the suppressive effect of IL-17A and IL-22 on HBV replication in the hepatocellular carcinoma cell line HepG2.2.15. IL-17A did not inhibit the proliferation of HepG2.2.15 cells. It decreased the levels of HBV s antigen (HBsAg) and HBV e antigen (HBeAg) in culture medium and the levels of intracellular HBV DNA. By contrast, blockage of IL-17 receptor (IL-17R) increased the levels of HBsAg and extracellular HBV DNA in culture medium and the levels of intracellular HBV DNA. The expression of antiviral proteins, including myxovirus resistance A (MxA) and oligoadenylate synthetase (OAS), was enhanced by IL-17A. IL-22 and anti-human IL-22 receptor (IL-22R) antibody did not change any indexes. We demonstrated that IL-17A effectively suppressed HBV replication in a noncytopathic manner and the over-expression of MxA and OAS mRNA was involved in the suppression of HBV replication by IL-17A.

MxA transcripts with distinct first exons and modulation of gene expression levels by single-nucleotide polymorphisms in human bronchial epithelial cells

Myxovirus resistance A (MxA) is a major interferon (IFN)-inducible antiviral protein. Promoter single-nucleotide polymorphisms (SNPs) of MxA near the IFN-stimulated response element (ISRE) have been frequently associated with various viral diseases, including emerging respiratory infections. We investigated the expression profile of MxA transcripts with distinct first exons in human bronchial epithelial cells. For primary culture, the bronchial epithelium was isolated from lung tissues with different genotypes, and total RNA was subjected to real-time reverse transcription polymerase chain reaction. The previously reported MxA transcript (T1) and a recently registered transcript with a distinct 5′ first exon (T0) were identified. IFN-β and polyinosinic–polycytidylic acid induced approximately 100-fold higher expression of the T1 transcript than that of the T0 transcript, which also had a potential ISRE motif near its transcription start site. Even without inducers, the T1 transcript accounted for approximately two thirds of the total expression of MxA, levels of which were significantly associated with its promoter and exon 1 SNPs (rs17000900, rs2071430, and rs464138). Our results suggest that MxA observed in respiratory viral infections is possibly dominated by the T1 transcript and partly influenced by relevant 5′ SNPs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-012-0663-8) contains supplementary material, which is available to authorized users.

Retinoic acid inducible gene-I (RIG-I) signaling of hepatic stellate cells inhibits hepatitis C virus replication in hepatocytes

Retinoic acid inducible gene-I (RIG-I) is critical in the activation of the type I IFN-dependent antiviral innate immune response to hepatitis C virus (HCV) infection. We examined whether hepatic stellate cells (HSC; LX-2) possess a functional RIG-I signaling pathway and produce antiviral factors that can inhibit HCV. We showed that LX-2 cells treated with the RIG-I ligand (5'ppp-dsRNA) expressed significantly higher levels of IFN-β and IFN-λ than the control cells. The RIG-I activation in LX-2 cells also induced the expression of Toll-like receptor 3 (TLR3) and IFN regulatory factor-7 (IRF-7), the key regulators of the IFN signaling pathway. When HCV Japanese fulminant hepatitis (JFH)-1-infected hepatocytes were co-cultured with LX-2 cells stimulated with 5'ppp-dsRNA or incubated in media conditioned with supernatant (SN) from 5'ppp-dsRNA-stimulated LX-2 cells, HCV replication in hepatocytes was suppressed significantly. This LX-2 cell action on HCV replication was mediated through both IFN-β and IFN-λ, as Abs to IFN-α/β or IFN-λ receptors could neutralize the LX-2 SN-mediated anti-HCV effect. The role of IFNs in LX-2 cell-mediated anti-HCV activity is further supported by the observation that LX-2 SN treatment induced the expression of IFN stimulated genes, 2'-5'-oligoadenylate synthase-1 (OAS-1) and myxovirus resistance A (MxA), in HCV-infected Huh7 cells. These observations highlight the importance of HSC in liver innate immunity against HCV infection via a RIG-I-mediated signaling pathway.

MxA expression induced by α‐defensin in healthy human periodontal tissue

Although periodontal tissue is continually challenged by microbial plaque, it is generally maintained in a healthy state. To understand the basis for this, we investigated innate antiviral immunity in human periodontal tissue. The expression of mRNA encoding different antiviral proteins, myxovirus resistance A (MxA), protein kinase R (PKR), oligoadenylate synthetase (OAS), and secretory leukocyte protease inhibitor (SLPI) were detected in both healthy tissue and that with periodontitis. Immunostaining data consistently showed higher MxA protein expression in the epithelial layer of healthy gingiva as compared with tissue with periodontitis. Human MxA is thought to be induced by type I and III interferons (IFNs) but neither cytokine type was detected in healthy periodontal tissues. Treatment in vitro of primary human gingival epithelial cells (HGECs) with α-defensins, but not with the antimicrobial peptides β-defensins or LL-37, led to MxA protein expression. α-defensin was also detected in healthy periodontal tissue. In addition, MxA in α-defensin-treated HGECs was associated with protection against avian influenza H5N1 infection and silencing of the MxA gene using MxA-targeted-siRNA abolished this antiviral activity. To our knowledge, this is the first study to uncover a novel pathway of human MxA induction, which is initiated by an endogenous antimicrobial peptide, namely α-defensin. This pathway may play an important role in the first line of antiviral defense in periodontal tissue.

The Influence of Polymorphisms in the MxA Promoter and the elF-2α Regulatory Region 2 on the Natural outcome of HBV Infection

Interferon(IFN) stimulates the expression of a number of genes encoding enzymes with antiviral activities, including myxovirus resistance A(MxA) and double-stranded RNA-dependent protein kinase(PKR). PKR is also activated by dsRNA, this leads to the phosphorylation of eukaryotic initiation factor 2a(elF-2α), which halts viral replication. We investigated whether polymorphisms in MxA promoter and elF-2α regulatory region 2(elF-2α reg2) influenced the natural outcome of hepatitis B virus(HBV) infection. A total of 243 patients with chronic HBV infection and 160 patients with self-limited HBV infection were used to genotype and identify this single-nucleotide polymorphism(SNP) by polymerase chain reaction-restriction fragment length polymorphism and sequencing, respectively. The distribution of the genotypes(GG, GT, and TT) at position -88 in the MxA promoter was 52.7%, 44.4%, and 2.9% in patients with chronic HBV infection and 41.3%, 43.1%, and 15.6% in patients with self-limited HBV infection, respectively. The frequencies of the TT genotype at position -88 in the MxA promoter were significantly higher among patients with self-limited HBV infection compared with patients with chronic HBV infection (odds ratio=6.24; 95% CI: 2.63-14.81; P=0.001). However, the polymorphisms both at position -123 in the MxA promoter and in the elF-2α reg2 were not significantly different between the two groups (P>0.05). In conclusion, Polymorphism at position -88G/T in the MxA promoter influences the natural outcomes of HBV infection to some extent. This SNP of MxA promoter may be used as a clinical prognostic marker of HBV infection.

Significance of the Myxovirus Resistance A (MxA) Gene −123C>A Single‐Nucleotide Polymorphism in Suppressed Interferon β Induction of Severe Acute Respiratory Syndrome Coronavirus Infection

Myxovirus resistance A (MxA) is an antiviral protein induced by interferon α and β (IFN-α, IFN-β) that can inhibit viral replication. The minor alleles of the -88G>T and -123C>A MxA promoter single-nucleotide polymorphisms (SNPs) are associated with increased promoter activity and altered response to IFN-α and IFN-β treatment. Here, we demonstrate that the 123A minor allele provided stronger binding affinity to nuclear proteins extracted from IFN-β-untreated cells than did the wild-type allele, whereas the -88T allele showed preferential binding after IFN-β stimulation. Endogenous IFN-α and IFN-β induction can be suppressed in severe acute respiratory syndrome (SARS) coronavirus infection. In support of our in vitro findings, a large case-control genetic-association study for SARS coronavirus infection confirmed that the -123A minor-allele carriers were significantly associated with lower risk of SARS coronavirus infection, whereas the -88T minorallele carriers were insignificant after adjustment for confounding effects. This suggests that -123C>A plays a more important role in modulating basal MxA expression, thus contributing more significantly to innate immune response against viral infections that suppress endogenous IFN-α and IFN-β induction such as SARS coronavirus.

Evaluation of an Attenuated Vesicular Stomatitis Virus Vector Expressing Interferon-β for Use in Malignant Pleural Mesothelioma: Heterogeneity in Interferon Responsiveness Defines Potential Efficacy

Abstract Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-beta (IFN-beta) gene (VSV.IFN-beta). We conducted this study to determine the ability of VSV.IFN-beta to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-beta did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-beta. In the eight resistant lines, pretreatment with IFN-beta prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-beta protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2',5'-oligo-adenylate-synthetase (2'5'-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-beta protein and no IFN- or VSV-induced changes in PKR, MxA, and 2'5'-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-beta by immunostaining for the presence of p48 protein.

Interferon-λ induces G1 phase arrest or apoptosis in oesophageal carcinoma cells and produces anti-tumour effects in combination with anti-cancer agents

Signal pathways of novel type III interferons (IFN-λs) are similar to those of type I IFNs (IFN-α/β) but their distinct functions have not been well characterised. We examined the growth suppressive activity of IFN-λ1 with nine human oesophageal carcinoma cell lines expressing the IFN-λ receptor complexes. Among them, three lines but not others showed IFN-λ1-mediated growth suppression by inducing G1 phase arrest or apoptosis. The G1 phase arrest was accompanied by the up-regulation of p21 and dephosphorylation of retinoblastoma (Rb), and the apoptosis was evidenced by cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). Similar but not identical susceptibility was found in IFN-α-treated oesophageal carcinoma cells. Despite the differential suppressive responses among the cells, all the cells increased the expression of the myxovirus resistance A (MxA) and 2′,5′-oligoadenylate synthetase (2′,5′-OAS) genes and class I antigens of the major histocompatibility complexes (MHC) with IFN-λ1 treatment. Fibroblasts and mesenchymal stem cells, positive for IFN-α receptor (IFNAR), lacked one of the IFN-λ receptor complexes and Het-1A, immortalised oesophageal epithelium cells, were insensible to the IFN-λ1-induced growth suppression. IFN-λ1 produced combinatory anti-tumour effects with chemotherapeutic agents, cisplatin (CDDP) and 5-fluorouracil (5-FU), in IFN-λ1-sensitive oesophageal carcinoma cells but not in normal or Het-1A cells, while IFN-α achieved the combinatory suppressive effects to normal cells. These data collectively show that IFN-λ1 responsiveness is tissue-specific due to the restricted receptors expression and is diversified even among cells of the same lineage, and suggest that IFN-λ1 is a potential therapeutic agent for oesophageal carcinoma without damaging surrounding tissues.

Interferon-Induced Expression of MxA in the Respiratory Tract of Rhesus Macaques Is Suppressed by Influenza Virus Replication

To determine the relationship between influenza A virus replication and innate antiviral immune responses, rhesus monkeys were given oseltamivir before influenza A/Memphis/7/01 (H1N1) challenge. We found that oseltamivir treatment significantly reduced viral replication in the trachea (p < 0.029). Further, in the trachea of both treated and untreated monkeys the mRNA levels of most innate antiviral molecules in the IFN-alphabeta pathway were dramatically increased by 24 h postinfection. However, the mRNA level of a single IFN-stimulated gene, MxA (myxovirus resistance A), the IFN-stimulated gene known to be critical in blocking influenza virus replication, was significantly lower in the tracheal lavages of untreated monkeys than in the oseltamivir-treated monkeys (p = 0.05). These results demonstrate for the first time that uncontrolled influenza A virus replication actively suppresses MxA gene expression and emphasize the critical role of innate immunity in controlling influenza virus replication in vivo.

Pharmacogenetics of MXA SNPs in interferon-β treated multiple sclerosis patients

The myxovirus resistance A ( MXA) mRNA has been extensively investigated for assessing the biologic responses of multiple sclerosis (MS) patients to interferon-β (IFN-β) therapy. The objective of this study was to evaluate the associations between two MXA promoter region single nucleotide polymorphisms (rs2071430 and rs17000900) and the gene expression responses, clinical and MRI phenotypes in IFN-β treated MS patients. The rs2071430 and rs17000900 SNPs, which are located in or near an interferon-stimulated response element (ISRE), were genotyped in 179 relapsing MS patients. Quantitative MRI measurements were available for 101 patients on IFN-β monotherapy. Gene expression was assessed in 22 anti-interferon-β neutralizing antibody negative patients. No significant association was found between the MXA genotype at these two SNPs and clinical, MRI and MXA gene expression in MS patients treated with IFN-β therapy.

Expression and secretion of antiviral factors by trophoblast cells following stimulation by the TLR-3 agonist, Poly(I : C)

During pregnancy, the placenta may become exposed to micro-organisms, such as viruses, which may pose a substantial threat to the embryo/fetus well-being. Recent insight into the immunological capabilities of the trophoblast suggests that the placenta may function as an active barrier by recognizing and responding to pathogens through Toll-like receptors (TLRs). The objective of this study was to determine whether the engagement of TLR-3 with viral dsRNA by first-trimester trophoblast could induce the production of factors necessary to generate an antiviral response. Therefore, trophoblast cells were exposed to the TLR-3 agonist, Poly(I : C). We report that following stimulation with Poly(I : C), first-trimester trophoblast cells produce interferon beta (IFNbeta) and secretory leukocyte protease inhibitor (SLPI), as well as the intracellular factors 2',5'-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). This response is TLR-3 specific because the TLR-4 ligand, lipopolysaccharide (LPS), had no effect on the production of these antimicrobial factors. Furthermore, we describe a positive feedback mechanism in which IFNbeta enhances the antiviral response by promoting the production of OAS, MxA and APOBEC3G. These findings suggest that trophoblast cells are able to recognize and specifically respond to viral products in a highly regulated fashion and that the placenta may be pivotal in the control of viral infections at the maternal-fetal interface.

Interferon-Stimulated Gene Expression in Black and White Hepatitis C Patients During Peginterferon Alfa-2a Combination Therapy

Black American patients are less likely to eradicate hepatitis C virus (HCV) infections during treatment with peginterferon (PEG-IFN) and ribavirin. We hypothesized that racial differences in IFN-stimulated antiviral gene induction during treatment might be responsible. We examined myxovirus resistance-A (MxA), RNA-dependent protein kinase (PKR), 2'-5' oligoadenylate synthetase (2,5-OAS), and adenosine deaminase-1 (ADAR1) gene expression in the peripheral blood mononuclear cells (PBMCs) of 31 black and 11 white HCV genotype 1 patients at baseline and at weeks 4-12 during PEG-IFN alfa-2a combination treatment. The primary study end point was the early virologic response (EVR)-either an undetectable serum HCV-RNA level or a > or =2-log decrease in serum HCV-RNA level at week 12 compared with week 0. The EVR rate was 67.7% in blacks and 63.6% in whites. Both blacks and whites experienced a significant (200%-500%) increase in 2,5-OAS, MxA, PKR, and ADAR1 expression at treatment weeks 4-12 compared with baseline (P < .01). However, the relationship between IFN-stimulated gene expression and the EVR differed by race. White responders exhibited higher 2,5-OAS and MxA levels at week 4 than white nonresponders (P < .05). IFN-stimulated gene levels did not correlate with EVR in blacks. Black responders had much lower MxA and PKR levels at week 4 than black nonresponders (P < .05). However, black responders maintained increased 2,5-OAS, MxA, and PKR levels from weeks 4-12, whereas the levels decreased to baseline at weeks 8-12 in black nonresponders. The mechanisms of resistance to PEG-IFN combination therapy may be different in black and white HCV genotype 1 patients.

Polymorphisms of interferon-inducible genes OAS-1 and MxA associated with SARS in the Vietnamese population

We hypothesized that host antiviral genes induced by type I interferons might affect the natural course of severe acute respiratory syndrome (SARS). We analyzed single nucleotide polymorphisms (SNPs) of 2′,5′-oligoadenylate synthetase 1 (OAS-1), myxovirus resistance-A (MxA), and double-stranded RNA-dependent protein kinase in 44 Vietnamese SARS patients with 103 controls. The G-allele of non-synonymous A/G SNP in exon 3 of OAS-1 gene showed association with SARS ( p = 0.0090). The G-allele in exon 3 of OAS-1 and the one in exon 6 were in strong linkage disequilibrium and both of them were associated with SARS infection. The GG genotype and G-allele of G/T SNP at position −88 in the MxA gene promoter were found more frequently in hypoxemic group than in non-hypoxemic group of SARS ( p = 0.0195). Our findings suggest that polymorphisms of two IFN-inducible genes OAS-1 and MxA might affect susceptibility to the disease and progression of SARS at each level.

SOCS-1 and SOCS-3 inhibit IFN-α-induced expression of the antiviral proteins 2,5-OAS and MxA

Although the use of IFN-α in combination with ribavirin has improved the treatment efficacy of chronic hepatitis C virus (HCV) infection, 20–50% of patients still fail to eradicate the virus depending on the HCV genotype. Recently, overexpression of HCV core protein has been shown to inhibit IFN signaling and induce SOCS-3 expression. Aim of this study was to examine the putative role of SOCS proteins in IFN resistance. By Western blot analysis, a 4-fold induction of STAT-1/3 phosphorylation by IFN-α was observed in mock-transfected HepG2 clones. In contrast, IFN-induced STAT-1/3 phosphorylation was considerably downregulated by SOCS-1/3 overexpression. In mock-transfected cells, IFN-α induced 2 ′,5 ′-OAS and myxovirus resistance A (MxA) promoter activity 40- to 80-fold and 10- to 35-fold, respectively, and this effect was abrogated in SOCS-1/3 overexpressing cells. As detected by Northern blot technique, IFN-α potently induced 2 ′,5 ′-OAS and MxA mRNA expression in the control clones. Overexpression of SOCS-1 completely abolished both 2 ′,5 ′-OAS and MxA mRNA expression, whereas SOCS-3 mainly inhibited 2 ′,5 ′-OAS mRNA expression. Our results demonstrate that SOCS-1 and SOCS-3 proteins inhibit IFN-α-induced activation of the Jak–STAT pathway and expression of the antiviral proteins 2 ′,5 ′-OAS and MxA. These data suggest a potential role of SOCS proteins in IFN resistance during antiviral treatment.